There are four known species of horseshoe crabs, Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda. The amebocyte is the only, or predominant, type of circulating cell in the blood of the horseshoe crab. The amebocyte is a nucleated cell having densely packed granules which contain endotoxin-sensitive clotting factors. Armstrong, in Biomedical Applications of the Horseshoe Crab (Limulidae), pages 73-93 (Alan R. Liss, Inc., New York, N.Y. 1979), describes the details of the active pseudopod-directed motility of Limulus polyphemus amebocyte migrating in vitro on glass microscope coverslips and in vivo in gill leaflets isolated from young animals. Although the important role of the amebocyte in the coagulation of the blood of Limulus has been known for many years, it was only recently established that the elements required for coagulation: a serine esterase of 150,000 apparent molecular weight requiring both Ca.sup.2+ and endotoxin for activation, and a clottable protein of 23,000-27,000 apparent molecular weight, are all intracellular.
The reaction between lysates of amebocytes and bacterial endotoxin provides the basis for the most sensitive currently available in vitro assay for endotoxin. Studies of hemolymph coagulation in the horseshoe crab, Limulus polyphemus, establish that the circulating blood cells, the amebocytes, secrete the blood clotting factors in response to bacterial endotoxins. Minute quantities of endotoxin cause amebocytes to aggregate and degranulate concomitant to clot formation, as described by Bang, Bull. Johns Hopkins Hosp. 98,325 (1956); and Levin and Bang, Bull. Johns Hopkins Hosp. 115, 337 (1964). All of the clotting proteins released by the amebocytes are contained within the numerous large granules filling the cytoplasm, as reported by Murer, et al., J. Cellular Physiol 86, 533 (1975). Bang reported an increase in free granules in the lymph of endotoxin injected crabs implying that stimulated amebocytes simply rupture (Bang, 1955). Dumont, et al., J. Morphol 119, 181 (1966) examined the ultrastructural changes of stimulated amebocytes and suggested that the granule membrane fused with the plasmalemma during release. Ornberg and Reese, pp. 125-130 in Biomedical Applications of the Horseshoe Crab Dec. 8 (1979), using a quick-freeze method, captured the membrane fusion in electron micrographs, demonstrating that this was the mechanism for release.
Although the production and application of the Limulus Amebocyte Lysate (LAL) test has become more standardized in recent years, significant variation occurs in lysate produced by different manufacturers and even from lot-to-lot within material produced by individual manufacturers. The successful cultivation of Limulus amebocytes in the laboratory would enable production of a lysate that is not subject to the variability of the natural environment. Such a standardized production would most probably reduce batch variability and seasonal variability of lysate.
In vitro cultivation of amebocytes might also eliminate or decrease the need to bleed horseshoe crabs so that the collection, transportation and possible depletion of the horseshoe crab population would not be necessary. Although the U.S. horseshoe crab population appears to be stable and in good numbers at this time, their number could easily be diminished by overutilization, as well as by diminished and deteriorating habitat. Unlike the American horseshoe crab which has apparently coped with twentieth century habitat and survived the challenge from the fertilizer, fishing and medical industries, its Japanese counterpart has not been so fortunate. As noted by Niwa, et al., Jap. J. Med. Sci. Biol., 27, 108 (1974), as quoted in Shishikura, et al., Biomedical Applications of the Horseshoe Crab (Limulidae), p 185-201 (Alan R Liss, Inc., N.Y., N.Y. 1979), the Japanese horseshoe crab is an "endangered species and seemingly on its way to extinction due to reclamations and contamination".
Although Limulus appears to be extremely resilient to being bled from the collection of amebocytes, the impact of bleeding activity on the horseshoe crab population has not been clearly documented in the literature. Proposed rules which wee released in the Federal Register on Aug. 11, 1978 underscore the potential problem by requesting manufacturers of LAL to guarantee that the production of LAL will not have an adverse impact on existing crab populations and that horseshoe crabs will be returned alive to their natural environment after a single collection of their blood.
Interest in the laboratory cultivation of Limulus cells and tissues is not new. As early as 1959, when cell culture was still emerging from its infancy, Sanborn and workers reported in Biol. Bull. 117, 399, on the successful in vitro cultivation of a number of cell types, other than amebocytes, from Limulus tissues. These included primary explants of ovary, hepatopancreas, nerve, leg and cardiac muscle, all of which were cultivated by use of a simple hanging drop method. Although the medium employed in this study was not named, it contained inorganic salts, multiple sugars and organic acids all of which are consistent with contemporary cell culture media, and five to ten percent sterile Limulus serus. Cells were maintained up to thirty days, with vacuolation and granulation becoming pronounced after ten days.
Pearson and Woodland reported in Biomedical Applications of the Horseshoe Crab (Limulidae), 93-102 (alan R. Liss, Inc., New York, N.Y. 1979), that the general morphology of cells cultivated from Limulus primary explants are not unlike those encountered in explants of vertebrate tissue. They selected and tested a number of common cell culture media for their potential use in the cultivation of Limulus amebocytes: Minimal Essential Medium (MEM), Leibovitz (L-15), Medium 199 and Grace's Insect Medium (GIM). Lysate preparations from cultured amebocytes were tested for potency by using graded endotoxin solutions ranging from 0.1 ng to 1000 ng per ml. Since solid clots were not formed by lysate preparations, a graded endpoint was used ranging from 0 (negative) to +3 (a clot that would run when the tube was tilted 180.degree.). Intermediate values wee based on degree of opacity, viscosity and production of floccules. None of the lysate preparations from amebocytes grown in vitro gave more than slightly positive results (+1 reaction) in the presence of 1000 ng of endotoxin. In U.S. Pat. No. 4,229,541 to Pearson disclosed a method for cultivating amebocytes in culture. However, although the results demonstrate that the amebocytes replicated in culture, it is now well known that mature amebocytes do not replicate. No source for new amebocytes has been described by Pearson or anyone else.
It is therefore an object of the present invention to provide a method and means to provide and for culturing amebocyte-producing tissue from horseshoe crabs in vitro.
It is a further object to provide a method and means for producing pyrogen-sensitive horseshoe crab lysate in large quantity by in vitro production of amebocytes.
It is a still further object of the present invention to provide a method and means for producing pyrogen-sensitive Limulus polyphemus lysate which is not dependent on harvesting of wild Limulus polyphemus.